The long-term goal of this research is to define the mechanisms responsible for the malignant hyperthermia syndrome. We have previously shown that a universal feature of RyR1 MH mutations is an increased resting myoplasmic free Ca2+ concentration ([Ca2+]i) and more recently that they are associated with an increased resting Na+ concentration ([Na+]i). We have also shown that sarcoplasmic reticulum (SR) leak is a factor in controlling myoplasmic Ca2+ concentration at rest working in concert with sarcolemmal channels, pumps and exchangers. Furthermore, because the amount of Ca2+ in internal stores is limited compared to the extracellular pool, after MH is triggered these same sarcolemmal channels, pumps and exchangers and perhaps others must be involved in the maintenance of the MH syndrome. Because of the observed increase in [Na+]i in MH muscles, TRPCs are the most likely candidates. Our immediate objectives are to use two mouse models that we have created, RyR1-R163C a mouse model of human MH, and CSQ1 null mice, which have an MH like phenotype, to study how mutations that alter intracellular Ca2+ homeostasis cause a fulminant MH response when exposed to volatile anesthetics. These models will allow us to test a unified general hypothesis applicable to any and all MH mutations regardless of the location of the mutation: MH is caused by conformational changes in RyR1 as a result of a mutation, or by conformational changes in RyR1 induced indirectly by a mutation in CaV1.1 or another protein closely associated with RyR1 (as demonstrated by an MH like phenotype in Casq1 null mice). A transformative concept to be investigated here is that a defect in signaling among Ca2+ release unit proteins leading to increased RyR1 leak is a common convergent pathway leading to all MH susceptibility. Hypothesis 1: MH susceptibility is the result of a conformational change in RyR1 caused either by a RyR1 mutation, or induced indirectly by mutations in other proteins closely associated with RyR1 which results in increased RyR1 SR Ca2+ leak and sarcolemmal Na+ and Ca2+ entry. Specific Aim 1. To determine the filling state of the SR and rate of SR Ca2+ leak in MH muscle fibers. Specific Aim 2. To determine the role of TRPCs in causing abnormalities in sarcolemmal Na+ and Ca2+ entry RyR1-R163C and CSQ1 null muscles and then to determine if skeletal muscle specific over-expression of a dominant negative non-conducting TRPC6 channel can modify RyR1-R163C's MH phenotype. Hypothesis 2: In addition to blocking RyR1 SR Ca2+ release, dantrolene abrogates the MH phenotype by modulating RyR1 SR Ca2+ leak and sarcolemmal Na+ and Ca2+ entry. Specific Aim 3. To determine the mechanisms by which dantrolene diminishes aberrant Ca2+ signaling. Successful completion of these specific Aims will provide a more comprehensive understanding of MH.